Many epithelial tissues such as mammary glands can self-renew continuously in adult life. During development and regeneration, a pool of epithelial stem/progenitor cells proliferate and differentiate to generate multiple cell types. The long-term goal of our research is to use a multidisciplinary approach to study both genetic and epigenetic mechanisms that control the formation, maintenance, and differentiation of these stem/progenitor cells. This proposal focuses on investigating the role of mouse pygopus 2 (Pygo2) gene, which encodes an epigenetic activator, using mammary stem/progenitor cells as a model system. The first specific aim is to identify critical downstream targets of Pygo2 in mammary stem/progenitor cells. Stem/progenitor cell-enriched populations will be prepared from mammary epithelia of normal and Pygo2-deficient animals for transcriptional profiling and RT-PCR analysis to identify Pygo2-responsive genes. In situ expression, chromatin immunoprecipitation and reporter assays will then be performed to test whether Pygo2 directly activates putative stem/progenitor cell-specific genes and whether Pygo2 increases histone H3 K4 trimetylation at these target loci. The second specific aim is to test the hypothesis that Pygo2 functions in mammary stem/progenitor cell self-renewal by binding to K4-di- and tri-methylated histone H3 (H3K4me2/3, active marks of chromatin) and SET domain histone H3 K4 methyltransferase (HMT) complexes. Wild-type and mutant Pygo2 proteins deficient in H3K4me2/3 and/or HMT binding will be tested in progenitor cell expansion assays. Tandem affinity purification and mass spectrometric protein identification will be performed to identify the specific HMT and its associating factors in mammary progenitor cells. The significance of the proposed studies are several-fold: 1) they will aid our understanding of how normal development and homeostasis of epithelial stem/progenitor cells is regulated at genetic and epigenetic levels;2) such knowledge will provide a framework for our thinking on epithelial cancers, as cancer cells are known to re-activate developmentally important genes in an adult setting;3) knowledge on novel molecular interactions involving Pygo2 may implicate possible targets for therapeutic intervention of epithelial diseases or cancers of a stem/progenitor cell origin.